Collagen extensively and massively exists in the extracellular matrix of connective tissues of animals. A research report (Moseley et al., 2004, Br J Dermatol 150:401-413) points out that collagen may function as a biological marker of disease activity or therapy prognosis. Another research report (Ruszczak, Z., 2003, Adv Drug Deliv Rev 55:1595-1611) points out that collagen scaffold may function as the regenerative environment of cells. Thus, how to accurately detect the quantity of collagen and estimate the rates of reconstruction and degradation of collagen becomes an important subject for the clinical application of collagen.
The conventional methods for quantitatively analyzing collagen include the colorimetric analysis (Stegemann et al., 1967, Clin Chim Acta 18:267-273; Moore, S., 1968, J Biol Chem 243:6281-6283), the high performance liquid chromatography (HPLC), the liquid chromatography tandem mass spectrometry (MS) (Ikeda et al., 1993, J Chromatogr 621:133-138; Kindt et al., 2000, Anal Biochem 283:71-76), and the enzyme linked immunosorbent assay (ELISA) (Bellon, G., 1985, Anal Biochem 150:188-202). However, the abovementioned methods are very complicated and expensive. Therefore, they are not ideal methods for identifying and analyzing collagen quantitatively.
Compared with the abovementioned methods, the capillary electrophoresis is a simpler method for analyzing collagen quantitatively (Deyl, Z & Adam, M, 1989, J Chromatogr 488:161-197; Novotna et al., 1996, J Chromatogr B Biomed Appl 681:77-82; Deyl et al., 1997, J Chromatogr B Biomed Sci Appl 689:181-194; Chalmers, et al., 1999, J Chromatogr Sci 37:443-447). The capillary electrophoresis covers both advantages of electrophoresis and chromatography and can be automated. Therefore, it has been widely used to analyze and identify molecules. In capillary electrophoresis, a voltage is applied to the sample containing different molecules inside a capillary, and different molecules are separated by different electrophoretic mobility and electro-osmotic flow thereof. The silanol groups on the inner wall of the capillary will be dissociated and slightly negatively charged in the solution inside the capillary, particularly in an acidic solution. The negative charges will attract the cations and make the cations distributed on the capillary. When a voltage is applied to the capillary, the cations attached to the inner wall of the capillary will be attracted to the negative pole. The aggregated cations result in viscosity, which will drag the entire solution inside the capillary toward the negative pole and cause a bulk flow of the solution, i.e. the so-called electroosmotic flow. Because of the electrophoretic mobility difference and the electroosmotic flow, the capillary electrophoresis method has a high resolution and a high separation effect. Further, the capillary electrophoresis needs only a small amount of sample because the capillary functions as the electrophoresis path.
In the conventional capillary electrophoresis technology for analyzing collagen quantitatively, the sample should be purified, extracted, and then processed with cyanogen bromide (CNBr) (Deyl, Z & Adam, M, 1989, J Chromatogr 488:161-197; Deyl et al., 1997, J Chromatogr B Biomed Sci Appl 689:181-194; Deyl et al. 1999, J Chromatography A, 852:325-336) or enzymes such as collagenase (Ivan Mik{hacek over ( )}s'ik et al. 2006, J Chromatography B, 841:3-13) to obtain polypeptide fragments. The cyanogen bromide enables the cracking reaction of the methionine on the amino acid sequences of protein, and the products of the cracking reaction are analyzed with the capillary electrophoresis method. The collagen cracking reaction is time-consuming and prolongs the process of the collagen analysis. The cyanogen bromide is a toxic material and needs processing and disposing carefully. After purified and extracted, the sample may be processed with enzyme. For example, Harada (Bull. Chem. Soc. Jpn. vol. 69, 1996, pp. 3575-3579) discloses a capillary electrophoresis method for quantitatively identifying the different polypeptides of the collagen. Harada uses commercial pepsin-solubilized collagens (PSCs) as samples, and further digested with pepsin to decrease the telopeptide region. Besides, Harada also discloses a capillary which has a non-charged layer (Brij 35). The Brij is a nonionic surfactant, and it would bind to the polypeptide to form a micelle without carrying any charge. The results of Harada show that a good peak separation for each polypeptide was achieved at pH 5.6-6.5 reflecting various residues of telopeptide. Further, the pretreatments, such as extraction and cracking, should inevitably reduce the total amount of collagen and thus affect the quantitative result of the capillary electrophoresis analysis. Another example is done by Eckhardt (Adam Eckhardt et al., 2004, Journal of Chromatography A, 1051:111-117) and he discloses a capillary electrophoretic system using alkylamines containing background electrolytes at acid pH. To be specific, Eckhardt discloses an analytical buffer with amines, and mix the analytical buffer with different peptides of collagens to form a mixed sample. After that, Eckhardt drives the mixed sample through the capillary and decreases the effects of electro-osmotic flow via the amines with positive charges result in separating the different peptides to measure each amount of peptides. This kind of process “dynamic coating” also can only measure each amount of different peptides with the inevitable reduction and errors on the quantity of total collagen. There are errors while accounting each amount of different peptides, so that when you sum the total amount of each peptide to calculate the total amount of collagen, there is a bigger error. Therefore, it is not accurate enough to measure total amount of collagen via the above methods.
In addition, there are some methods without cracking the native collagen such as the method from Zhang (Jing Zhang et al., 2004, Electrophoresis 25, 3416-3421). Zhang discloses a quantitative measurement of methylated collagen by capillary electrophoresis which proposes an index to quantify the degree of collagen methylation that also correlates with their effects on cell proliferation. Zhang uses a polyvinyl alcohol-coated fused-silica capillary and a phosphate buffer having 0.05% hydroxypropylmthylcellulose to quantify the collagens in which their carboxylic groups were esterified to obtain methylated collagens. In the results of Zhang, the methylated collagens were separated into different peaks using the phosphate buffer with 0.05% hydroxypropylmthylcellulose. Even if Zhang did not crack collagens as the analyte, Zhang can only quantify different methylated collagen respectively with his esterification pretreatment and separation condition rather than quantify the total amount of collagen.
With regard to the novelty, obviousness, and industrial application, an optimal condition for capillary electrophoresis method to identify collagen as a single peak in the chromatogram is required without pretreatment, extraction, purification or further processing the collagen containing sample. Yet, the single peak in the chromatogram can offer an accurate quantification way for total collagen.